DNA Barcode Data Accurately Assign Higher Spider Taxa

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DNA barcode data accurately assign higher spider taxa

Jonathan A. Coddington1, Ingi Agnarsson1,2, Ren-Chung Cheng3, Klemen Čandek3, Amy Driskell1, Holger Frick4, Matjaž Gregorič3, Rok Kostanjšek5, Christian Kropf4, Matthew Kweskin1, Tjaša Lokovšek3, Miha Pipan3,6, Nina Vidergar3 and Matjaž Kuntner1,3

1 National Museum of Natural History, Smithsonian Institution, Washington, D.C., United States 2 Department of Biology, University of Vermont, Burlington, Vermont, United States 3 EZ Lab, Institute of Biology, Research Centre of the Slovenian Academy of Sciences and Arts, Ljubljana, Slovenia 4 Department of Invertebrates, Natural History Museum Bern, Bern, Switzerland 5 Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia 6 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

ABSTRACT The use of unique DNA sequences as a method for taxonomic identification is no longer fundamentally controversial, even though debate continues on the best markers, methods, and technology to use. Although both existing databanks such as GenBank and BOLD, as well as reference taxonomies, are imperfect, in best case scenarios ‘‘barcodes’’ (whether single or multiple, organelle or nuclear, loci) clearly are an increasingly fast and inexpensive method of identification, especially as compared to manual identification of unknowns by increasingly rare expert taxonomists. Because most species on Earth are undescribed, a complete reference database at the species level is impractical in the near term. The question therefore arises whether unidentified species can, using DNA barcodes, be accurately assigned to more inclusive groups such as genera and families—taxonomic ranks of putatively monophyletic groups for which the global inventory is more complete and stable. We used a carefully chosen test library of CO1 sequences from 49 families, 313 genera, and 816 species of spiders to assess the accuracy of genus and family-level assignment. We used BLAST queries of Submitted 11 January 2016 each sequence against the entire library and got the top ten hits. The percent sequence Accepted 10 June 2016 identity was reported from these hits (PIdent, range 75–100%). Accurate assignment Published 20 July 2016 of higher taxa (PIdent above which errors totaled less than 5%) occurred for genera at Corresponding author PIdent values >95 and families at PIdent values ≥ 91, suggesting these as heuristic Matjaž Kuntner, [email protected] thresholds for accurate generic and familial identifications in spiders. Accuracy of Academic editor identification increases with numbers of species/genus and genera/family in the library; Sven Rahmann above five genera per family and fifteen species per genus all higher taxon assignments Additional Information and were correct. We propose that using percent sequence identity between conventional Declarations can be found on barcode sequences may be a feasible and reasonably accurate method to identify animals page 21 to family/genus. However, the quality of the underlying database impacts accuracy of DOI 10.7717/peerj.2201 results; many outliers in our dataset could be attributed to taxonomic and/or sequencing Copyright errors in BOLD and GenBank. It seems that an accurate and complete reference 2016 Coddington et al. library of families and genera of life could provide accurate higher level taxonomic Distributed under identifications cheaply and accessibly, within years rather than decades. Creative Commons CC-BY 4.0

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How to cite this article Coddington et al. (2016), DNA barcode data accurately assign higher spider taxa. PeerJ 4:e2201; DOI 10.7717/peerj.2201 Subjects Biodiversity, Bioinformatics, Ecology, Genetics, Taxonomy Keywords Taxonomic impediment, Family, Genus, Global Genome Initiative, Genome, DNA barcoding

INTRODUCTION Accurate identification of biological specimens has always limited the application of biological data to important societal problems. Obstacles are well-known and difficult: the vast majority of species are undescribed scientifically (Erwin, 1982; May, 1992; Mora et al., 2011); some unknown but large fraction of higher taxa are not monophyletic (Goloboff et al., 2009; Pyron & Wiens, 2011); many species can only be identified if certain life stages are available, e.g., adults (Coddington & Levi, 1991), classical data sources such as morphology imperfectly track species identity; the discipline of taxonomy continues to dwindle (Agnarsson & Kuntner, 2007); the classical process of taxonomic identification is mostly manual and cannot scale to provide the amounts of data required for real-time decisions such as environmental monitoring, invasive species, climate change, etc. DNA sequence data potentially can eliminate most of these obstacles. DNA barcoding uses a fragment of the mitochondrial gene cytochrome c oxidase subunit I (CO1) as a unique species diagnosis/identification tool in the animal kingdom (Hebert et al., 2003), with analogous single to several locus protocols applied for vascular plants, ferns, mosses, algae and fungi (Saunders, 2005; Kress & Erickson, 2007; Nitta, 2008; Chase & Fay, 2009; Liu et al., 2010), protists (Scicluna, Tawari & Clark, 2006), and prokaryotes (Barraclough et al., 2009). Due to relative ease and inexpensive sequencing, DNA barcoding is a popular tool in species identification and taxonomic applications (e.g., Doña et al., 2015; Xu et al., 2015; see also Collins & Cruickshank, 2013), and the method is no longer fundamentally controversial at the species level (Pentinsaari, Hebert & Mutanen, 2014; Lopardo & Uhl, 2014; Čandek & Kuntner, 2015; Anslan & Tedersoo, 2015; Wang et al., 2015). While most species remain undescribed, the situation is not so dire for larger monophyletic groups such as clades accorded the Linnaean ranks of genus or family. In assessing the state of knowledge about biodiversity, it is important to distinguish between the first scientific discovery of an exemplar of a lineage, and phylogenetic understanding of that lineage. Phylogenetic understanding—both tree topology and consequent taxonomic changes, are research programs with no clear end in sight. Linnaean rank is partially arbitrary, and one expects that the number of higher taxa will probably increase over time as understanding improves. Discovery, however, can have an objective definition: the year of the earliest formal taxonomic description of a member of the lineage or taxonomic group in which it is currently included. By this definition the earliest possible discovery of an animal lineage is 1758 (Linnaeus, 1758), or in the case of spiders, 1757 (Clerck, 1757). More illuminating are the latest discoveries of lineages with the rank of family within larger clades, because the data tell us something about progress towards broad scale knowledge of biodiversity. The species representing the most recent discovery of a family of birds, for example, is the Broad-billed Sapayoa, Sapayoa aenigma Hunt, 1903 (Sapayoaidae). The species representing the most recently discovered mammal family is Kitti’s hog-nosed bat, Craseonycteris thonglongyai Hill, 1974 (Craseonycteridae). For flowering plants, it is

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 2/25 Figure 1 First discovery of major clades of life. Accumulation curve of dates of first discovery (year of first description of a contained species) of families for six major clades of life, 1758–2010.

Gomortega keule (Molina) Baill, 1972 (Gomertegaceae). For bees, it is Stenotritus elegans Smith, 1853 (Stenotritidae). For spiders, a megadiverse and poorly known group, it is Trogloraptor marchingtoni Griswold, Audisio & Ledford, 2012 (Trogloraptoridae), but the second most recent discovery of an unambiguously new spider family was in 1955, Gradungulidae (Forster, 1955). Figure 1 illustrates the tempo of first discovery of families for these five well-known clades. At the family level, these curves are essentially asymptotic, implying that science is close to completing the inventory of clades ranked as families for these large lineages. On the other hand, for Bacteria and Archaea (Fig. 1), as one would expect, the curve is not asymptotic at all but sharply increasing; prokaryote discovery and understanding is obviously just beginning. In fact, although many new eukaryote families are named every year, the vast majority of these new names result from advances in phylogenetic understanding, not biological discovery of major new forms of life. The last ten years of Zoological Record suggests that roughly 5–10 truly new families are discovered per year. In the context of the above question—approximate taxonomic assignment of organisms using DNA sequences—these data suggest that our knowledge of major clades of life is approaching completion. The Global Genome Initiative (GGI; http://ggi.si.edu/) of the Smithsonian Institution via the GGI Knowledge Portal (http://ggi.eol.org/) has tabulated a complete list of families of life, which total 9,650—on the whole a surprisingly small number. 10,000 barcodes, more or less, seems like a feasible goal. If we were able to assemble a complete database of DNA sequences at the family level, would it suffice to identify any eukaryote on Earth to the family level? While the literature on species identification success of DNA barcodes comprises thousands of studies, only a few have tested their effectiveness at the level of higher

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 3/25 taxonomic units. In the seminal paper on DNA barcodes, Hebert et al. (2003) established that animal CO1 sequences can roughly assign taxa to phyla (96% success) or orders (100% success). However, their test was based on a neighbor joining tree-building approach, and it remained unknown if sequence data itself, i.e., percent identity among taxa, can be used in this way. Similarly, Nagy et al. (2012) showed that DNA barcoding in reptiles usually correctly assigned barcodes to species, genus and family. Their approach was phylogenetic: they tested whether including a sequence in tree building rendered the higher group non-monophyletic, which would imply failure. Finally, Wilson et al. (2011) provided a similar tree based test in sphingid moths, and established reliabilities of correct generic and subfamily taxonomic assignments between 74 and 90% using a liberal, and only 66–84% using a strict, tree-based criterion. These authors argued that tree-based methods perform better than sequence comparison methods, but that reliability, of course, depends on the library completeness. Our project not only contributes original DNA barcode data for Central European spiders, but also works in synergy with the GGI towards a permanent preservation of genomic biodiversity: the formation of a collection of deeply frozen spider tissues and their DNA. We provide: (1) cryo-preserved tissues of reliably identified species of Central European spiders, and their vouchers photographed and deposited in public museums; (2) permanently frozen genomic DNA of these species; (3) publicly accessible DNA barcodes for these species (genetic sequence of cytochrome oxidase I—CO1) as public identification tool (Hebert et al., 2003) to facilitate organism identification, taxonomy, ecology and conservation. In addition, this project addresses to what extent higher level taxonomic units can be reliably identified using barcodes of unknown spiders, and specifically asks what percent sequence identity in BLAST results is necessary to correctly identify unknown taxa to the Linnaean genus and/or family. Other methods for classification of higher-level taxonomies such as RDP (Wang et al., 2007), UTAX (Edgar, 2010) and MEGAN (Huson et al., 2007) have primarily been developed for studies of microorganisms, using genetic markers for these groups, but less is known about using the CO1 barcoding gene in metazoans. We examine empirical data from Araneae barcode data to ask what is the percent sequence identity value above which 5% or less of higher level (genus/family) taxonomic identifications are incorrect and the extent to which frequency of correct identifications correlated with the number of taxa in this dataset, as would be expected given the dependence of BLAST on the reference database.

MATERIALS & METHODS Specimen processing and imaging We used automated and manual sampling methods for collecting spiders in the field in numerous localities in Slovenia and Switzerland. Faunistic and sampling details are published elsewhere (Čandek et al., 2013; see also 2015 corrigendum). Collected spiders were fixed in absolute ethanol immediately after being caught and the ethanol was replaced on the following day. Spiders were frozen at −80 ◦C, same day, or as soon as possible. In the

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 4/25 laboratory they were identified, labeled, photographed and processed for DNA extraction and sequencing (Čandek et al., 2013; see also 2015 corrigendum). Voucher specimens (voucher codes starting with 0078) are deposited at National Museum of Natural History, Smithsonian Institution (Washington D.C., USA), with duplicates (voucher codes starting with ARA) at Naturhistorisches Museum der Burgergemeinde Bern (Switzerland) and EZ LAB, ZRC SAZU (Ljubljana, Slovenia). Voucher images are published along with their barcodes (see Table 1) at http://ezlab.zrc- sazu.si/dna. All original sequences generated by this project have been submitted to BOLD systems, and those that BOLD accepted were also submitted to GenBank (Table 1). Tissues After specimen identification and processing, up to four legs (or in the case of very small individuals the whole prosoma) of a spider were removed and stored in fresh absolute ethanol in cryovials. Part of the tissue was used for DNA isolation while the other part remains permanently frozen at −80 ◦C at GGI facilities. The maintenance and use of these materials abides by the international legal standards and conventions of the biological genetic heritage (The Access and Benefit Sharing agreement as part of the 2010 Nagoya protocol). Molecular procedures At Laboratories of Analytical Biology (National Museum of Natural History, Smithsonian Institution, hereafter LAB), specimens were extracted using the AutoGenPrep phenol- chloroform automated extractor (AutoGen). Samples were digested overnight in buffer containing proteinase-k before extraction. At EZ Lab, specimens were extracted using the Mag MAXTM Express magnetic particle processor Type 700 with DNA Multisample kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocols with modifications (Vidergar, Toplak & Kuntner, 2014). At EZ Lab PCR was carried out using mainly primers LCO1490 and HCO2198 (Folmer et al., 1994). Standard reaction volume was 35 µL containing 2.3 mM MgCl2 (Promega), 0.15 mM each dNTP (Biotools), 0.4 µM of each primer, 0.2 µL 10 mg/mL BSA (Promega), 0.2 µL GoTaqFlexi polymerase (Promega) and 2 µL DNA. PCR cycling conditions were as follows: an initial denaturation step of 2 min at 94 ◦C followed by 35 cycles of 40 s at 94 ◦C, 1 min at 48 ◦–52 ◦C, 1 min at 72 ◦C, with final extension at 72 ◦C for 3 min. Additional primers were used for PCR for a few problematic specimens: dgLCO1490 and dgHCO2198 (Meyer & Paulay, 2005) and the reverse primer Chelicerate-R2 (Barrett & Hebert, 2005). Cycling parameters for difficult specimens were: 20 cycles of usual cycling protocol (above) followed by 15 cycles of 1.5 min at 94 ◦C, 1.5 min at 52 ◦C and 2 min at 72 ◦Cm version 5.6.6 (Kearse et al., 2012). EZ Lab PCR products were sent to be Sanger sequenced at Macrogen Inc. (Amsterdam, Netherlands), and the sequences were aligned, checked for sequencing errors and trimmed to match the barcode region in Geneious Pro version 5.6.6 (Kearse et al., 2012). At LAB, PCR was carried out using the primer pair LCO1490 (Folmer et al., 1994) and Chelicerate-R2 (Barrett & Hebert, 2005). A 10 µL reaction mix contained 2.5 mM MgCl2

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 5/25 Table 1 Original sequences this project submitted to BOLD and GenBank (only those on GenBank are also publically available on BOLD, for all others, see http://ezlab.zrc-sazu.si/dna/).

Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Agelenidae Agelena labyrinthica 00786574 SPSLO002-12 MNH, SI SVN Agelenidae Agelena labyrinthica ARA0239 SPSLO369-13 EZ LAB SVN Agelenidae Allagelena gracilens 00786557 SPSLO001-12 KX039062 MNH, SI SVN Agelenidae Coelotes terrestris 00786563 SPSLO003-12 KX039130 MNH, SI SVN Agelenidae Histopona torpida 00786599 SPSLO004-12 KX039207 MNH, SI SVN Agelenidae Histopona torpida ARA0063 SPSLO339-13 KX039208 EZ LAB SVN Agelenidae Inermocoelotes anoplus 00786586 SPSLO005-12 KX039220 MNH, SI SVN Agelenidae Inermocoelotes anoplus ARA0339 SPSLO392-13 KX039219 EZ LAB SVN Agelenidae Malthonica silvestris 00786304 SPSLO283-13 KX039239 MNH, SI SVN Agelenidae Malthonica silvestris ARA0427 SPSLO468-13 KX039238 EZ LAB SVN Agelenidae Tegenaria atrica 00786583 SPSLO006-12 KX039170 MNH, SI SVN Agelenidae Tegenaria atrica ARA0076 SPSLO341-13 KX039169 EZ LAB SVN Amaurobiidae Amaurobius erberi 00786571 SPSLO007-12 KX039070 MNH, SI SVN Amaurobiidae Amaurobius erberi ARA0120 SPSLO347-13 KX039069 EZ LAB SVN Amaurobiidae Amaurobius fenestralis 00786389 SPSLO189-12 KX039071 MNH, SI CHE Amaurobiidae Amaurobius ferox 00786307 SPSLO284-13 KX039072 MNH, SI SVN Amaurobiidae Amaurobius jugorum 00786585 SPSLO008-12 KX039073 MNH, SI SVN Anyphaenidae Anyphaena accentuata 00786584 SPSLO009-12 KX039076 MNH, SI SVN Anyphaenidae Anyphaena sabina 00786551 SPSLO010-12 KX039077 MNH, SI SVN Araneidae Aculepeira ceropegia 00786570 SPSLO011-12 KX039041 MNH, SI SVN Araneidae Aculepeira ceropegia ARA0355 SPSLO405-13 KX039040 NMBE CHE Araneidae Agalenatea redii 00786368 SPSLO095-12 KX039043 MNH, SI SVN Araneidae Agalenatea redii ARA0381 SPSLO429-13 KX039042 EZ LAB SVN Araneidae Araneus alsine 00786568 SPSLO012-12 KX039079 MNH, SI SVN Araneidae Araneus angulatus 00786552 SPSLO013-12 KX039081 MNH, SI SVN Araneidae Araneus angulatus ARA0001 SPSLO326-13 KX039080 EZ LAB SVN Araneidae Araneus diadematus 00786593 SPSLO014-12 KX039083 MNH, SI SVN Araneidae Araneus diadematus ARA0050 SPSLO336-13 KX039082 EZ LAB SVN Araneidae Araneus marmoreus 00786575 SPSLO015-12 KX039085 MNH, SI SVN Araneidae Araneus marmoreus ARA0030 SPSLO329-13 KX039084 EZ LAB SVN Araneidae Araneus quadratus 00786572 SPSLO016-12 KX039086 MNH, SI SVN Araneidae Araneus quadratus ARA0198 SPSLO362-13 KX039087 NMBE CHE Araneidae Araneus sturmi 00786561 SPSLO017-12 KX039089 MNH, SI SVN Araneidae Araneus sturmi ARA0108 SPSLO345-13 KX039088 EZ LAB SVN Araneidae Araniella cucurbitina 00786596 SPSLO018-12 KX039090 MNH, SI SVN Araneidae Araniella opisthographa ARA0393 SPSLO440-13 KX039091 EZ LAB SVN Araneidae Argiope bruennichi 00786589 SPSLO019-12 KX039093 MNH, SI SVN Araneidae Argiope bruennichi ARA0048 SPSLO335-13 KX039094 EZ LAB SVN (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 6/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Araneidae Cercidia prominens 00786498 SPSLO021-12 KX039116 MNH, SI SVN Araneidae Cercidia prominens 00786577 SPSLO020-12 KX039114 MNH, SI SVN Araneidae Cercidia prominens ARA0356 SPSLO406-13 KX039115 EZ LAB SVN Araneidae Cyclosa conica 00786573 SPSLO022-12 KX039135 MNH, SI SVN Araneidae Cyclosa conica ARA0380 SPSLO428-13 KX039136 NMBE CHE Araneidae Gibbaranea bituberculata 00786579 SPSLO023-12 KX039186 MNH, SI SVN Araneidae Gibbaranea bituberculata ARA0350 SPSLO400-13 KX039187 EZ LAB SVN Araneidae Hypsosinga albovittata 00786323 SPSLO191-12 KX039211 MNH, SI CHE Araneidae Hypsosinga pygmaea 00786555 SPSLO024-12 KX039212 MNH, SI SVN Araneidae Hypsosinga sanguinea 00786314 SPSLO285-13 KX039213 MNH, SI SVN Araneidae Hypsosinga sanguinea ARA0370 SPSLO419-13 KX039214 NMBE CHE Araneidae Larinioides sclopetarius 00786382 SPSLO096-12 KX039222 MNH, SI SVN Araneidae Leviellus thorelli 00786591 SPSLO025-12 KX039229 MNH, SI SVN Araneidae Leviellus thorelli ARA0353 SPSLO403-13 KX039228 EZ LAB SVN Araneidae Mangora acalypha 00786590 SPSLO026-12 KX039242 MNH, SI SVN Araneidae Mangora acalypha ARA0107 SPSLO344-13 KX039240 EZ LAB SVN Araneidae Mangora acalypha ARA0357 SPSLO407-13 KX039241 EZ LAB SVN Araneidae Neoscona adianta 00786330 SPSLO192-12 KX039282 MNH, SI SVN Araneidae Nuctenea umbratica 00786594 SPSLO027-12 KX039293 MNH, SI SVN Araneidae Nuctenea umbratica ARA0387 SPSLO435-13 KX039292 NMBE CHE Araneidae Parazygiella montana 00786582 SPSLO028-12 KX039307 MNH, SI SVN Araneidae Parazygiella montana ARA0354 SPSLO404-13 KX039308 NMBE CHE Araneidae Singa nitidula 00786597 SPSLO029-12 KX039376 MNH, SI SVN Araneidae Stroemiellus stroemi ARA0169 SPSLO358-13 KX039383 EZ LAB SVN Araneidae Zilla diodia 00786481 SPSLO097-12 KX039446 MNH, SI SVN Araneidae Zilla diodia ARA0342 SPSLO393-13 KX039447 EZ LAB SVN Araneidae Zygiella x-notata 00786326 SPSLO193-12 KX039450 MNH, SI CHE Atypidae Atypus piceus 00786580 SPSLO031-12 KX039096 MNH, SI SVN Atypidae Atypus piceus ARA0174 SPSLO359-13 KX039097 EZ LAB SVN Clubionidae Clubiona germanica 00786566 SPSLO032-12 KX039122 MNH, SI SVN Clubionidae Clubiona kulczynskii 00786404 SPSLO194-12 KX039123 MNH, SI CHE Clubionidae Clubiona neglecta 00786558 SPSLO033-12 KX039124 MNH, SI SVN Clubionidae Clubiona pseudoneglecta 00786286 SPSLO286-13 KX039125 MNH, SI SVN Clubionidae Clubiona reclusa 00786378 SPSLO195-12 KX039126 MNH, SI CHE Clubionidae Clubiona reclusa ARA0371 SPSLO420-13 KX039127 NMBE CHE Clubionidae Clubiona terrestris 00786457 SPSLO098-12 KX039129 MNH, SI SVN Clubionidae Clubiona terrestris ARA0242 SPSLO371-13 KX039128 EZ LAB SVN Corinnidae Phrurolithus minimus 00786559 SPSLO034-12 KX039341 MNH, SI SVN Dictynidae Argenna subnigra 00786283 SPSLO288-13 MNH, SI SVN Dictynidae Cicurina cicur 00786548 SPSLO035-12 KX039121 MNH, SI SVN Dictynidae Dictyna arundinacea 00786369 SPSLO196-12 KX039140 MNH, SI CHE Dictynidae Dictyna arundinacea ARA0379 SPSLO427-13 KX039139 NMBE CHE (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 7/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Dictynidae Dictyna civica 00786511 SPSLO036-12 KX039141 MNH, SI SVN Dictynidae Dictyna uncinata 00786345 SPSLO197-12 KX039143 MNH, SI SVN Dictynidae Dictyna uncinata ARA0423 SPSLO466-13 KX039142 EZ LAB SVN Dictynidae Lathys humilis 00786473 SPSLO198-12 KX039224 MNH, SI SVN Dictynidae Lathys humilis 00786550 SPSLO037-12 KX039223 MNH, SI SVN Dysderidae Dasumia canestrinii 00786581 SPSLO038-12 KX039137 MNH, SI SVN Dysderidae Dysdera adriatica 00786287 SPSLO289-13 KX039154 MNH, SI SVN Dysderidae Dysdera adriatica 00786296 SPSLO290-13 KX039155 MNH, SI SVN Dysderidae Dysdera ninnii ARA0244 SPSLO373-13 KX039156 EZ LAB SVN Filistatidae Filistata insidiatrix 00786560 SPSLO040-12 KX039181 MNH, SI SVN Filistatidae Filistata insidiatrix ARA0122 SPSLO348-13 KX039182 EZ LAB SVN Gnaphosidae Aphantaulax cincta 00786470 SPSLO199-12 KX039078 MNH, SI SVN Gnaphosidae Callilepis schuszteri 00786553 SPSLO041-12 KX039103 MNH, SI SVN Gnaphosidae Callilepis schuszteri ARA0333 SPSLO386-13 KX039102 EZ LAB SVN Gnaphosidae Drassodes lapidosus 00786505 SPSLO099-12 KX039150 MNH, SI SVN Gnaphosidae Drassodes pubescens 00786273 SPSLO291-13 KX039151 MNH, SI SVN Gnaphosidae Drassyllus villicus 00786556 SPSLO042-12 KX039153 MNH, SI SVN Gnaphosidae Drassyllus villicus ARA0337 SPSLO390-13 KX039152 EZ LAB SVN Gnaphosidae Gnaphosa bicolor 00786276 SPSLO292-13 KX039188 MNH, SI SVN Gnaphosidae Haplodrassus silvestris 00786578 SPSLO043-12 KX039196 MNH, SI SVN Gnaphosidae Micaria aenea 00786384 SPSLO200-12 KX039258 MNH, SI CHE Gnaphosidae Micaria pulicaria 00786274 SPSLO293-13 KX039259 MNH, SI SVN Gnaphosidae Nomisia exornata 00786564 SPSLO044-12 KX039291 MNH, SI SVN Gnaphosidae Phaeocedus braccatus 00786592 SPSLO045-12 KX039330 MNH, SI SVN Gnaphosidae Scotophaeus scutulatus 00786576 SPSLO046-12 KX039369 MNH, SI SVN Gnaphosidae Scotophaeus scutulatus ARA0082 SPSLO343-13 KX039370 EZ LAB SVN Gnaphosidae Trachyzelotes pedestris 00786279 SPSLO294-13 KX039419 MNH, SI SVN Gnaphosidae Zelotes apricorum 00786278 SPSLO295-13 KX039441 MNH, SI SVN Gnaphosidae Zelotes latreillei 00786540 SPSLO047-12 KX039443 MNH, SI SVN Gnaphosidae Zelotes latreillei ARA0191 SPSLO360-13 KX039442 EZ LAB SVN Gnaphosidae Zelotes subterraneus 00786588 SPSLO048-12 KX039445 MNH, SI CHE Gnaphosidae Zelotes subterraneus ARA0156 SPSLO355-13 KX039444 NMBE CHE Hahniidae Antistea elegans 00786405 SPSLO201-12 KX039075 MNH, SI CHE Hahniidae Antistea elegans ARA0384 SPSLO432-13 KX039074 NMBE CHE Hahniidae Hahnia difficilis 00786363 SPSLO202-12 KX039195 MNH, SI CHE Hahniidae Hahnia difficilis ARA0399 SPSLO445-13 KX039194 NMBE CHE Linyphiidae Agnyphantes expunctus 00786328 SPSLO203-12 KX039044 MNH, SI CHE Linyphiidae Agnyphantes expunctus ARA0429 SPSLO470-13 KX039045 NMBE CHE Linyphiidae Agyneta affinis 00786439 SPSLO115-12 KX039049 MNH, SI CHE Linyphiidae Agyneta affinis ARA0245 SPSLO374-13 KX039048 NMBE CHE Linyphiidae Agyneta alpica 00786443 SPSLO116-12 KX039050 MNH, SI CHE Linyphiidae Agyneta cauta 00786426 SPSLO204-12 KX039052 MNH, SI CHE (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 8/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Linyphiidae Agyneta cauta ARA0225 SPSLO367-13 KX039051 NMBE CHE Linyphiidae Agyneta conigera 00786448 SPSLO100-12 KX039053 MNH, SI CHE Linyphiidae Agyneta fuscipalpa 00786425 SPSLO218-12 MNH, SI CHE Linyphiidae Agyneta fuscipalpa ARA0268 SPSLO378-13 NMBE CHE Linyphiidae Agyneta gulosa 00786464 SPSLO219-12 KX039054 MNH, SI CHE Linyphiidae Agyneta innotabilis 00786393 SPSLO220-12 KX039055 MNH, SI CHE Linyphiidae Agyneta orites 00786419 SPSLO221-12 KX039057 MNH, SI CHE Linyphiidae Agyneta orites ARA0403 SPSLO449-13 KX039056 NMBE CHE Linyphiidae Agyneta rurestris 00786411 SPSLO117-12 KX039058 MNH, SI CHE Linyphiidae Agyneta rurestris ARA0419 SPSLO462-13 KX039059 EZ LAB SVN Linyphiidae Agyneta saxatilis 00786277 SPSLO298-13 KX039060 MNH, SI SVN Linyphiidae Agyneta simplicitarsis 00786295 SPSLO299-13 KX039061 MNH, SI SVN Linyphiidae Bolyphantes alticeps 00786465 SPSLO205-12 MNH, SI CHE Linyphiidae Bolyphantes luteolus 00786397 SPSLO101-12 KX039101 MNH, SI CHE Linyphiidae Bolyphantes luteolus ARA0214 SPSLO366-13 KX039100 NMBE CHE Linyphiidae Caracladus avicula 00786474 SPSLO206-12 KX039104 MNH, SI CHE Linyphiidae Caracladus avicula ARA0231 SPSLO368-13 KX039105 NMBE CHE Linyphiidae Caracladus zamoniensis 00786441 SPSLO102-12 KX039106 MNH, SI CHE Linyphiidae Centromerus pabulator 00786451 SPSLO207-12 KX039108 MNH, SI CHE Linyphiidae Centromerus pabulator ARA0421 SPSLO464-13 KX039107 NMBE CHE Linyphiidae Centromerus subalpinus 00786412 SPSLO208-12 KX039110 MNH, SI CHE Linyphiidae Centromerus subalpinus ARA0250 SPSLO375-13 KX039109 NMBE CHE Linyphiidae Ceratinella brevipes 00786317 SPSLO234-12 KX039112 MNH, SI CHE Linyphiidae Ceratinella brevipes 00786450 SPSLO103-12 KX039113 MNH, SI CHE Linyphiidae Ceratinella brevipes ARA0363 SPSLO413-13 KX039111 NMBE CHE Linyphiidae Diplocephalus crassilobus 00786294 SPSLO296-13 KX039144 MNH, SI SVN Linyphiidae Diplocephalus latifrons 00786461 SPSLO209-12 KX039145 MNH, SI CHE Linyphiidae Diplostyla concolor 00786533 SPSLO049-12 KX039146 MNH, SI SVN Linyphiidae Drapetisca socialis 00786587 SPSLO050-12 KX039149 MNH, SI SVN Linyphiidae Drapetisca socialis ARA0405 SPSLO451-13 KX039148 EZ LAB SVN Linyphiidae Entelecara acuminata 00786460 SPSLO210-12 KX039164 MNH, SI CHE Linyphiidae Erigone atra ARA0257 SPSLO377-13 KX039171 NMBE CHE Linyphiidae Erigone dentipalpis ARA0256 SPSLO376-13 KX039172 NMBE CHE Linyphiidae Erigone remota 00786416 SPSLO107-12 KX039174 MNH, SI CHE Linyphiidae Erigonella ignobilis ARA0164 SPSLO357-13 KX039173 NMBE CHE Linyphiidae Floronia bucculenta 00786545 SPSLO051-12 KX039183 MNH, SI SVN Linyphiidae Frontinellina frutetorum 00786567 SPSLO052-12 KX039184 MNH, SI SVN Linyphiidae Frontinellina frutetorum ARA0441 SPSLO480-13 KX039185 EZ LAB SVN Linyphiidae Gonatium hilare 00786565 SPSLO053-12 KX039189 MNH, SI SVN Linyphiidae Gonatium rubellum 00786318 SPSLO212-12 KX039191 MNH, SI CHE Linyphiidae Gonatium rubellum ARA0386 SPSLO434-13 KX039190 NMBE CHE Linyphiidae Gonatium rubens 00786331 SPSLO213-12 KX039192 MNH, SI CHE (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 9/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Linyphiidae Gonatium rubens ARA0358 SPSLO408-13 KX039193 NMBE CHE Linyphiidae Improphantes nitidus 00786449 SPSLO109-12 KX039218 MNH, SI CHE Linyphiidae Incestophantes frigidus ARA0211 SPSLO364-13 NMBE CHE Linyphiidae Kaestneria dorsalis 00786598 SPSLO054-12 KX039221 MNH, SI SVN Linyphiidae Lepthyphantes leprosus 00786342 SPSLO214-12 KX039225 MNH, SI SVN Linyphiidae Lepthyphantes nodifer ARA0433 SPSLO473-13 KX039226 NMBE CHE Linyphiidae Linyphia hortensis 00786526 SPSLO112-12 KX039230 MNH, SI SVN Linyphiidae Linyphia hortensis ARA0397 SPSLO443-13 KX039231 NMBE CHE Linyphiidae Linyphia triangularis 00786547 SPSLO056-12 KX039232 MNH, SI SVN Linyphiidae Linyphia triangularis ARA0004 SPSLO327-13 KX039233 EZ LAB SVN Linyphiidae Macrargus rufus ARA0213 SPSLO365-13 KX039237 NMBE CHE Linyphiidae Mansuphantes fragilis 00786415 SPSLO114-12 KX039243 MNH, SI CHE Linyphiidae Mansuphantes fragilis ARA0276 SPSLO380-13 KX039244 NMBE CHE Linyphiidae Maso sundevalli 00786400 SPSLO216-12 KX039248 MNH, SI CHE Linyphiidae Maso sundevalli ARA0360 SPSLO410-13 KX039247 NMBE CHE Linyphiidae Megalepthyphantes collinus 00786569 SPSLO057-12 KX039249 MNH, SI SVN Linyphiidae Mermessus trilobatus 00786395 SPSLO118-12 KX039250 MNH, SI SVN Linyphiidae Metopobactrus prominulus 00786437 SPSLO119-12 KX039257 MNH, SI CHE Linyphiidae Micrargus alpinus ARA0270 SPSLO379-13 KX039260 NMBE CHE Linyphiidae Micrargus herbigradus 00786466 SPSLO223-12 KX039261 MNH, SI CHE Linyphiidae Microctenonyx subitaneus 00786463 SPSLO224-12 KX039262 MNH, SI CHE Linyphiidae Microlinyphia impigra 00786350 SPSLO228-12 KX039263 MNH, SI CHE Linyphiidae Microlinyphia impigra ARA0369 SPSLO418-13 KX039264 NMBE CHE Linyphiidae Microlinyphia pusilla 00786417 SPSLO225-12 KX039265 MNH, SI CHE Linyphiidae Minicia marginella 00786371 SPSLO120-12 KX039267 MNH, SI SVN Linyphiidae Minicia marginella ARA0410 SPSLO455-13 KX039268 NMBE CHE Linyphiidae Minyriolus pusillus ARA0285 SPSLO382-13 KX039269 NMBE CHE Linyphiidae Mughiphantes cornutus ARA0372 SPSLO421-13 KX039272 NMBE CHE Linyphiidae Mughiphantes mughi 00786319 SPSLO227-12 KX039274 MNH, SI CHE Linyphiidae Mughiphantes mughi 00786322 SPSLO217-12 KX039275 MNH, SI CHE Linyphiidae Mughiphantes mughi ARA0361 SPSLO411-13 KX039273 NMBE CHE Linyphiidae Mughiphantes mughi ARA0411 SPSLO456-13 KX039276 NMBE CHE Linyphiidae Nematogmus sanguinolentus 00786490 SPSLO162-12 KX039279 MNH, SI SVN Linyphiidae Nematogmus sanguinolentus ARA0359 SPSLO409-13 KX039278 NMBE CHE Linyphiidae Neriene clathrata ARA0352 SPSLO402-13 KX039287 EZ LAB SVN Linyphiidae Neriene furtiva 00786471 SPSLO229-12 KX039289 MNH, SI SVN Linyphiidae Neriene furtiva ARA0145 SPSLO353-13 KX039288 EZ LAB SVN Linyphiidae Neriene radiata ARA0152 SPSLO354-13 KX039290 NMBE CHE Linyphiidae Obscuriphantes obscurus 00786354 SPSLO231-12 KX039295 MNH, SI CHE Linyphiidae Obscuriphantes obscurus ARA0407 SPSLO453-13 KX039294 NMBE CHE Linyphiidae Oedothorax gibbifer 00786396 SPSLO232-12 KX039296 MNH, SI CHE Linyphiidae Oryphantes angulatus ARA0398 SPSLO444-13 NMBE CHE (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 10/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Linyphiidae Ostearius melanopygius 00786339 SPSLO122-12 KX039297 MNH, SI SVN Linyphiidae Palliduphantes pallidus 00786341 SPSLO233-12 KX039302 MNH, SI CHE Linyphiidae Panamomops tauricornis ARA0375 SPSLO424-13 KX039303 NMBE CHE Linyphiidae Pityohyphantes phrygianus 00786316 SPSLO236-12 KX039351 MNH, SI CHE Linyphiidae Pityohyphantes phrygianus ARA0347 SPSLO397-13 KX039352 NMBE CHE Linyphiidae Pocadicnemis juncea 00786421 SPSLO237-12 KX039354 MNH, SI CHE Linyphiidae Pocadicnemis juncea ARA0409 SPSLO454-13 KX039355 NMBE CHE Linyphiidae Pocadicnemis pumila 00786422 SPSLO238-12 KX039356 MNH, SI CHE Linyphiidae Porrhomma pallidum 00786410 SPSLO239-12 KX039357 MNH, SI CHE Linyphiidae Porrhomma pygmaeum 00786292 SPSLO301-13 MNH, SI SVN Linyphiidae Scotinotylus alpigena 00786444 SPSLO125-12 KX039367 MNH, SI CHE Linyphiidae Scotinotylus alpigena ARA0163 SPSLO356-13 KX039366 NMBE CHE Linyphiidae Scotinotylus clavatus 00786420 SPSLO240-12 KX039368 MNH, SI CHE Linyphiidae Silometopus elegans 00786409 SPSLO126-12 KX039373 MNH, SI CHE Linyphiidae Tapinocyba affinis 00786406 SPSLO127-12 KX039387 MNH, SI CHE Linyphiidae Tapinocyba affinis ARA0362 SPSLO412-13 KX039386 NMBE CHE Linyphiidae Tenuiphantes alacris 00786343 SPSLO241-12 KX039389 MNH, SI CHE Linyphiidae Tenuiphantes alacris ARA0420 SPSLO463-13 KX039388 NMBE CHE Linyphiidae Tenuiphantes cristatus 00786305 SPSLO302-13 KX039390 MNH, SI CHE Linyphiidae Tenuiphantes cristatus ARA0418 SPSLO461-13 KX039391 NMBE CHE Linyphiidae Tenuiphantes flavipes 00786528 SPSLO060-12 KX039392 MNH, SI SVN Linyphiidae Tenuiphantes flavipes ARA0336 SPSLO389-13 KX039393 NMBE CHE Linyphiidae Tenuiphantes jacksoni 00786356 SPSLO242-12 MNH, SI CHE Linyphiidae Tenuiphantes jacksoni 00786430 SPSLO128-12 MNH, SI CHE Linyphiidae Tenuiphantes jacksoni ARA0435 SPSLO475-13 NMBE CHE Linyphiidae Tenuiphantes jacksonoides ARA0374 SPSLO423-13 KX039394 NMBE CHE Linyphiidae Tenuiphantes mengei 00786301 SPSLO300-13 KX039396 MNH, SI CHE Linyphiidae Tenuiphantes mengei 00786413 SPSLO243-12 KX039397 MNH, SI CHE Linyphiidae Tenuiphantes mengei ARA0415 SPSLO459-13 KX039395 NMBE CHE Linyphiidae Tenuiphantes tenebricola 00786418 SPSLO244-12 KX039398 MNH, SI CHE Linyphiidae Tenuiphantes tenebricola ARA0414 SPSLO458-13 KX039399 NMBE CHE Linyphiidae Tenuiphantes tenuis 00786383 SPSLO129-12 MNH, SI SVN Linyphiidae Tiso aestivus ARA0422 SPSLO465-13 KX039413 NMBE CHE Linyphiidae Tiso vagans 00786351 SPSLO246-12 KX039414 MNH, SI CHE Linyphiidae Tiso vagans ARA0401 SPSLO447-13 KX039415 NMBE CHE Linyphiidae Walckenaeria antica 00786429 SPSLO130-12 KX039421 MNH, SI CHE Linyphiidae Walckenaeria furcillata 00786431 SPSLO131-12 KX039422 MNH, SI CHE Liocranidae Agroeca brunnea 00786320 SPSLO247-12 KX039046 MNH, SI SVN Liocranidae Agroeca brunnea ARA0392 SPSLO439-13 KX039047 EZ LAB SVN Liocranidae Liocranum rupicola 00786516 SPSLO061-12 KX039234 MNH, SI SVN Liphistiidae Liphistius sp ARA0240 SPSLO482-15 KX039235 EZ LAB MYS Lycosidae Alopecosa accentuata 00786365 SPSLO248-12 MNH, SI CHE (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 11/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Lycosidae Alopecosa pulverulenta 00786527 SPSLO063-12 KX039064 MNH, SI SVN Lycosidae Alopecosa pulverulenta ARA0349 SPSLO399-13 KX039063 NMBE CHE Lycosidae Alopecosa sulzeri 00786452 SPSLO249-12 KX039065 MNH, SI SVN Lycosidae Alopecosa taeniata 00786538 SPSLO062-12 KX039066 MNH, SI CHE Lycosidae Alopecosa trabalis 00786509 SPSLO064-12 KX039067 MNH, SI SVN Lycosidae Alopecosa trabalis ARA0438 SPSLO478-13 KX039068 EZ LAB SVN Lycosidae Arctosa fulvolineata 00786336 SPSLO250-12 MNH, SI SVN Lycosidae Arctosa lutetiana 00786407 SPSLO132-12 MNH, SI SVN Lycosidae Arctosa maculata 00786312 SPSLO305-13 KX039092 MNH, SI SVN Lycosidae Aulonia albimana 00786524 SPSLO133-12 KX039099 MNH, SI SVN Lycosidae Aulonia albimana ARA0338 SPSLO391-13 KX039098 EZ LAB SVN Lycosidae Hogna radiata 00786502 SPSLO065-12 KX039210 MNH, SI SVN Lycosidae Hogna radiata ARA0368 SPSLO417-13 KX039209 EZ LAB SVN Lycosidae Pardosa agrestis 00786385 SPSLO134-12 KX039309 MNH, SI SVN Lycosidae Pardosa amentata 00786337 SPSLO251-12 KX039311 MNH, SI SVN Lycosidae Pardosa amentata ARA0413 SPSLO457-13 KX039310 NMBE CHE Lycosidae Pardosa bifasciata 00786453 SPSLO252-12 KX039312 MNH, SI SVN Lycosidae Pardosa blanda 00786358 SPSLO253-12 KX039314 MNH, SI CHE Lycosidae Pardosa blanda ARA0345 SPSLO396-13 KX039313 NMBE CHE Lycosidae Pardosa cf. lugubris 00786529 SPSLO066-12 KX039316 MNH, SI CHE Lycosidae Pardosa cf. lugubris ARA0065 SPSLO340-13 KX039315 EZ LAB SVN Lycosidae Pardosa ferruginea 00786309 SPSLO306-13 KX039317 MNH, SI CHE Lycosidae Pardosa hortensis 00786289 SPSLO307-13 KX039318 MNH, SI SVN Lycosidae Pardosa oreophila 00786310 SPSLO308-13 KX039319 MNH, SI CHE Lycosidae Pardosa oreophila 00786321 SPSLO254-12 KX039320 MNH, SI CHE Lycosidae Pardosa oreophila ARA0348 SPSLO398-13 KX039321 NMBE CHE Lycosidae Pardosa palustris 00786514 SPSLO067-12 KX039323 MNH, SI SVN Lycosidae Pardosa palustris ARA0406 SPSLO452-13 KX039322 NMBE CHE Lycosidae Pardosa proxima 00786311 SPSLO309-13 KX039324 MNH, SI SVN Lycosidae Pardosa riparia 00786315 SPSLO310-13 KX039326 MNH, SI SVN Lycosidae Pardosa riparia ARA0243 SPSLO372-13 KX039325 NMBE CHE Lycosidae Pirata piraticus 00786375 SPSLO255-12 KX039346 MNH, SI CHE Lycosidae Pirata piraticus ARA0430 SPSLO471-13 KX039347 NMBE CHE Lycosidae Piratula hygrophila 00786388 SPSLO135-12 KX039348 MNH, SI SVN Lycosidae Piratula knorri 00786402 SPSLO136-12 MNH, SI SVN Lycosidae Trochosa spinipalpis 00786344 SPSLO137-12 MNH, SI SVN Lycosidae Trochosa spinipalpis ARA0388 SPSLO436-13 EZ LAB SVN Lycosidae Xerolycosa nemoralis 00786541 SPSLO068-12 KX039424 MNH, SI CHE Lycosidae Xerolycosa nemoralis ARA0335 SPSLO388-13 KX039423 NMBE CHE Mimetidae Ero furcata 00786390 SPSLO256-12 KX039175 MNH, SI CHE Miturgidae Cheiracanthium erraticum 00786367 SPSLO138-12 KX039117 MNH, SI SVN Miturgidae Cheiracanthium mildei 00786355 SPSLO139-12 KX039118 MNH, SI SVN (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 12/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Miturgidae Cheiracanthium punctorium 00786519 SPSLO140-12 KX039120 MNH, SI SVN Miturgidae Cheiracanthium punctorium ARA0056 SPSLO337-13 KX039119 EZ LAB SVN Nemesiidae Nemesia pannonica 00786333 SPSLO311-13 KX039280 MNH, SI SVN Philodromidae Philodromus albidus 00786272 SPSLO312-13 KX039332 MNH, SI SVN Philodromidae Philodromus aureolus 00786539 SPSLO069-12 KX039333 MNH, SI SVN Philodromidae Philodromus cespitum 00786513 SPSLO070-12 KX039335 MNH, SI CHE Philodromidae Philodromus cespitum ARA0400 SPSLO446-13 KX039334 EZ LAB SVN Philodromidae Philodromus dispar 00786492 SPSLO142-12 KX039336 MNH, SI SVN Philodromidae Philodromus praedatus 00786500 SPSLO071-12 KX039338 MNH, SI SVN Philodromidae Philodromus praedatus ARA0404 SPSLO450-13 KX039337 NMBE CHE Philodromidae Philodromus pulchellus 00786475 SPSLO072-12 KX039339 MNH, SI SVN Philodromidae Philodromus pulchellus ARA0344 SPSLO395-13 KX039340 EZ LAB SVN Philodromidae Philodromus vagulus 00786366 SPSLO257-12 MNH, SI CHE Philodromidae Philodromus vagulus ARA0351 SPSLO401-13 NMBE CHE Philodromidae Thanatus formicinus 00786530 SPSLO073-12 KX039403 MNH, SI SVN Philodromidae Tibellus macellus 00786493 SPSLO074-12 KX039412 MNH, SI SVN Philodromidae Tibellus macellus ARA0334 SPSLO387-13 KX039411 EZ LAB SVN Pholcidae Psilochorus simoni 00786501 SPSLO076-12 KX039359 MNH, SI SVN Pisauridae Pisaura mirabilis 00786487 SPSLO144-12 KX039349 MNH, SI SVN Pisauridae Pisaura mirabilis ARA0383 SPSLO431-13 KX039350 NMBE CHE Salticidae Evarcha arcuata 00786332 SPSLO259-12 KX039177 MNH, SI CHE Salticidae Evarcha arcuata ARA0062 SPSLO338-13 KX039178 EZ LAB SVN Salticidae Evarcha falcata 00786408 SPSLO145-12 MNH, SI SVN Salticidae Evarcha falcata ARA0037 SPSLO331-13 KX039179 EZ LAB SVN Salticidae Evarcha jucunda 00786503 SPSLO077-12 KX039180 MNH, SI SVN Salticidae Evarcha michailovi 00786313 SPSLO313-13 MNH, SI SVN Salticidae Evarcha michailovi 00786458 SPSLO260-12 MNH, SI SVN Salticidae Evarcha michailovi ARA0436 SPSLO476-13 EZ LAB SVN Salticidae Hasarius adansoni 00786348 SPSLO261-12 KX039197 MNH, SI SVN Salticidae Heliophanus aeneus 00786293 SPSLO314-13 MNH, SI SVN Salticidae Heliophanus auratus 00786282 SPSLO315-13 KX039198 MNH, SI SVN Salticidae Heliophanus cupreus 00786518 SPSLO146-12 KX039199 MNH, SI SVN Salticidae Heliophanus cupreus ARA0382 SPSLO430-13 KX039200 NMBE CHE Salticidae Heliophanus flavipes 00786510 SPSLO147-12 KX039202 MNH, SI SVN Salticidae Heliophanus flavipes ARA0396 SPSLO442-13 KX039201 EZ LAB SVN Salticidae Heliophanus kochii 00786495 SPSLO078-12 KX039203 MNH, SI SVN Salticidae Icius subinermis 00786381 SPSLO148-12 KX039217 MNH, SI SVN Salticidae Leptorchestes berolinensis 00786512 SPSLO079-12 KX039227 MNH, SI SVN Salticidae Macaroeris nidicolens 00786338 SPSLO262-12 KX039236 MNH, SI SVN Salticidae Marpissa muscosa 00786523 SPSLO080-12 KX039245 MNH, SI SVN Salticidae Marpissa nivoyi 00786496 SPSLO081-12 KX039246 MNH, SI SVN Salticidae Myrmarachne formicaria 00786432 SPSLO149-12 KX039277 MNH, SI SVN (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 13/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Salticidae Neon reticulatus 00786370 SPSLO150-12 KX039281 MNH, SI SVN Salticidae Pellenes seriatus 00786462 SPSLO263-12 KX039329 MNH, SI SVN Salticidae Pellenes seriatus 00786504 SPSLO082-12 KX039327 MNH, SI SVN Salticidae Pellenes seriatus ARA0439 SPSLO479-13 KX039328 EZ LAB SVN Salticidae Philaeus chrysops 00786472 SPSLO264-12 KX039331 MNH, SI SVN Salticidae Pseudeuophrys lanigera 00786280 SPSLO316-13 KX039358 MNH, SI SVN Salticidae Saitis barbipes 00786507 SPSLO083-12 KX039363 MNH, SI SVN Salticidae Salticus scenicus 00786362 SPSLO265-12 KX039364 MNH, SI CHE Salticidae Sibianor aurocinctus 00786377 SPSLO266-12 MNH, SI CHE Salticidae Sibianor aurocinctus ARA0385 SPSLO433-13 NMBE CHE Salticidae Sitticus rupicola 00786525 SPSLO084-12 KX039377 MNH, SI CHE Salticidae Sitticus rupicola ARA0378 SPSLO426-13 KX039378 NMBE CHE Scytodidae Scytodes thoracica 00786521 SPSLO085-12 KX039371 MNH, SI SVN Segestriidae Segestria senoculata 00786281 SPSLO317-13 KX039372 MNH, SI SVN Sparassidae Micrommata virescens 00786497 SPSLO086-12 MNH, SI SVN Sparassidae Micrommata virescens ARA0365 SPSLO414-13 KX039266 NMBE CHE Tetragnathidae Metellina mengei 00786536 SPSLO087-12 KX039251 MNH, SI CHE Tetragnathidae Metellina mengei ARA0373 SPSLO422-13 KX039252 NMBE CHE Tetragnathidae Metellina merianae 00786298 SPSLO318-13 KX039253 MNH, SI CHE Tetragnathidae Metellina merianae ARA0394 SPSLO441-13 KX039254 EZ LAB SVN Tetragnathidae Metellina segmentata 00786357 SPSLO152-12 KX039255 MNH, SI SVN Tetragnathidae Metellina segmentata ARA0431 SPSLO472-13 KX039256 EZ LAB SVN Tetragnathidae Pachygnatha degeeri 00786399 SPSLO153-12 KX039300 MNH, SI SVN Tetragnathidae Tetragnatha nigrita 00786534 SPSLO088-12 KX039400 MNH, SI SVN Tetragnathidae Tetragnatha nigrita ARA0041 SPSLO332-13 KX039401 EZ LAB SVN Tetragnathidae Tetragnatha pinicola 00786361 SPSLO267-12 KX039402 MNH, SI CHE Tetragnathidae Tetragnatha pinicola 00786520 SPSLO155-12 MNH, SI SVN Theridiidae Asagena phalerata 00786346 SPSLO156-12 KX039095 MNH, SI SVN Theridiidae Crustulina guttata 00786454 SPSLO268-12 KX039132 MNH, SI SVN Theridiidae Crustulina guttata ARA0437 SPSLO477-13 KX039131 EZ LAB SVN Theridiidae Crustulina scabripes 00786479 SPSLO089-12 KX039134 MNH, SI SVN Theridiidae Crustulina scabripes ARA0137 SPSLO352-13 KX039133 EZ LAB SVN Theridiidae Dipoena melanogaster 00786506 SPSLO090-12 KX039147 MNH, SI SVN Theridiidae Enoplognatha afrodite 00786532 SPSLO157-12 KX039160 MNH, SI SVN Theridiidae Enoplognatha afrodite ARA0135 SPSLO350-13 KX039159 EZ LAB SVN Theridiidae Enoplognatha latimana 00786329 SPSLO269-12 KX039161 MNH, SI CHE Theridiidae Enoplognatha ovata 00786515 SPSLO158-12 KX039163 MNH, SI SVN Theridiidae Enoplognatha ovata ARA0367 SPSLO416-13 KX039162 NMBE CHE Theridiidae Episinus angulatus 00786386 SPSLO159-12 KX039165 MNH, SI SVN Theridiidae Episinus maculipes 00786488 SPSLO160-12 KX039166 MNH, SI SVN Theridiidae Episinus truncatus 00786327 SPSLO270-12 KX039168 MNH, SI CHE Theridiidae Episinus truncatus ARA0132 SPSLO349-13 KX039167 EZ LAB SVN (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 14/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Theridiidae Euryopis flavomaculata 00786468 SPSLO271-12 KX039176 MNH, SI SVN Theridiidae Heterotheridion nigrovariegatum 00786482 SPSLO161-12 KX039206 MNH, SI SVN Theridiidae Heterotheridion nigrovariegatum ARA0343 SPSLO394-13 KX039205 EZ LAB SVN Theridiidae Neottiura bimaculata 00786445 SPSLO163-12 KX039284 MNH, SI SVN Theridiidae Neottiura bimaculata ARA0366 SPSLO415-13 KX039283 NMBE CHE Theridiidae Neottiura herbigrada 00786467 SPSLO272-12 KX039285 MNH, SI SVN Theridiidae Neottiura suaveolens 00786427 SPSLO164-12 KX039286 MNH, SI SVN Theridiidae Paidiscura pallens 00786288 SPSLO319-13 KX039301 MNH, SI SVN Theridiidae Parasteatoda lunata 00786476 SPSLO165-12 KX039304 MNH, SI SVN Theridiidae Parasteatoda tepidariorum 00786531 SPSLO091-12 KX039305 MNH, SI SVN Theridiidae Parasteatoda tepidariorum ARA0329 SPSLO384-13 KX039306 EZ LAB SVN Theridiidae Phylloneta impressa 00786401 SPSLO273-12 KX039342 MNH, SI CHE Theridiidae Phylloneta impressa ARA0428 SPSLO469-13 KX039343 NMBE CHE Theridiidae Phylloneta sisyphia 00786364 SPSLO274-12 KX039344 MNH, SI CHE Theridiidae Phylloneta sisyphia ARA0416 SPSLO460-13 KX039345 NMBE CHE Theridiidae Platnickina tincta 00786380 SPSLO167-12 KX039353 MNH, SI SVN Theridiidae Robertus lividus ARA0201 SPSLO363-13 KX039360 NMBE CHE Theridiidae Robertus mediterraneus 00786334 SPSLO275-12 MNH, SI CHE Theridiidae Robertus mediterraneus 00786433 SPSLO168-12 MNH, SI CHE Theridiidae Robertus scoticus 00786290 SPSLO320-13 MNH, SI SVN Theridiidae Robertus truncorum 00786435 SPSLO169-12 KX039361 MNH, SI CHE Theridiidae Robertus truncorum ARA0280 SPSLO381-13 KX039362 NMBE CHE Theridiidae Sardinidion blackwalli 00786271 SPSLO321-13 KX039365 MNH, SI SVN Theridiidae Simitidion simile 00786549 SPSLO170-12 KX039375 MNH, SI SVN Theridiidae Simitidion simile ARA0442 SPSLO481-13 KX039374 EZ LAB SVN Theridiidae Steatoda bipunctata 00786325 SPSLO276-12 KX039380 MNH, SI CHE Theridiidae Steatoda bipunctata ARA0029 SPSLO328-13 KX039379 EZ LAB SVN Theridiidae Steatoda triangulosa 00786489 SPSLO171-12 KX039382 MNH, SI SVN Theridiidae Steatoda triangulosa ARA0046 SPSLO334-13 KX039381 EZ LAB SVN Theridiidae Theridion betteni 00786340 SPSLO277-12 KX039404 MNH, SI CHE Theridiidae Theridion pinastri 00786480 SPSLO172-12 KX039406 MNH, SI SVN Theridiidae Theridion pinastri ARA0136 SPSLO351-13 KX039405 EZ LAB SVN Theridiidae Theridion varians 00786374 SPSLO173-12 KX039408 MNH, SI SVN Theridiidae Theridion varians ARA0043 SPSLO333-13 KX039407 EZ LAB SVN Thomisidae Diaea livens 00786359 SPSLO174-12 KX039138 MNH, SI SVN Thomisidae Ebrechtella tricuspidata 00786508 SPSLO092-12 KX039157 MNH, SI SVN Thomisidae Ebrechtella tricuspidata ARA0033 SPSLO330-13 KX039158 EZ LAB SVN Thomisidae Heriaeus hirtus 00786469 SPSLO278-12 KX039204 MNH, SI SVN Thomisidae Misumena vatia 00786387 SPSLO175-12 KX039270 MNH, SI SVN Thomisidae Misumena vatia ARA0081 SPSLO342-13 KX039271 EZ LAB SVN Thomisidae Ozyptila atomaria 00786522 SPSLO176-12 KX039298 MNH, SI CHE Thomisidae Ozyptila nigrita 00786499 SPSLO093-12 KX039299 MNH, SI SVN (continued on next page)

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 15/25 Table 1 (continued) Family Genus Species Sample ID Process ID GenBank Voucher Collected in accession stored at number Thomisidae Synema globosum 00786485 SPSLO177-12 KX039384 MNH, SI SVN Thomisidae Synema globosum ARA0390 SPSLO438-13 KX039385 NMBE CHE Thomisidae Thomisus onustus 00786455 SPSLO280-12 KX039410 MNH, SI SVN Thomisidae Thomisus onustus ARA0426 SPSLO467-13 KX039409 EZ LAB SVN Thomisidae Tmarus piger 00786484 SPSLO178-12 KX039417 MNH, SI SVN Thomisidae Tmarus piger ARA0376 SPSLO425-13 KX039418 EZ LAB SVN Thomisidae Xysticus acerbus 00786483 SPSLO179-12 KX039425 MNH, SI SVN Thomisidae Xysticus audax 00786347 SPSLO180-12 KX039427 MNH, SI SVN Thomisidae Xysticus audax ARA0402 SPSLO448-13 KX039426 EZ LAB SVN Thomisidae Xysticus bifasciatus 00786543 SPSLO181-12 KX039428 MNH, SI SVN Thomisidae Xysticus cristatus 00786537 SPSLO182-12 KX039430 MNH, SI SVN Thomisidae Xysticus cristatus ARA0389 SPSLO437-13 KX039429 NMBE CHE Thomisidae Xysticus desidiosus 00786372 SPSLO183-12 MNH, SI SVN Thomisidae Xysticus erraticus 00786275 SPSLO322-13 KX039431 MNH, SI SVN Thomisidae Xysticus kempeleni 00786486 SPSLO184-12 KX039432 MNH, SI SVN Thomisidae Xysticus kochi 00786303 SPSLO323-13 KX039433 MNH, SI SVN Thomisidae Xysticus kochi ARA0434 SPSLO474-13 KX039434 EZ LAB SVN Thomisidae Xysticus lanio 00786477 SPSLO185-12 KX039435 MNH, SI SVN Thomisidae Xysticus lineatus 00786535 SPSLO186-12 KX039437 MNH, SI SVN Thomisidae Xysticus lineatus ARA0304 SPSLO383-13 KX039436 EZ LAB SVN Thomisidae Xysticus macedonicus 00786376 SPSLO281-12 KX039438 MNH, SI CHE Thomisidae Xysticus tenebrosus 00786478 SPSLO187-12 KX039440 MNH, SI SVN Thomisidae Xysticus tenebrosus ARA0332 SPSLO385-13 KX039439 EZ LAB SVN Titanoecidae Titanoeca tristis 00786297 SPSLO324-13 KX039416 MNH, SI SVN Uloboridae Hyptiotes paradoxus 00786546 SPSLO188-12 KX039216 MNH, SI SVN Uloboridae Hyptiotes paradoxus ARA0241 SPSLO370-13 KX039215 EZ LAB SVN Uloboridae Uloborus walckenaerius 00786324 SPSLO282-12 KX039420 MNH, SI SVN Zoridae Zora spinimana 00786494 SPSLO094-12 KX039449 MNH, SI SVN Zoridae Zora spinimana ARA0192 SPSLO361-13 KX039448 NMBE CHE

Notes. MNH, SI, National Museum of Natural History, Smithsonian Institution; EZ LAB, Evolutionary Zoology Lab; ZRC SAZU; NMBE, Naturhistorisches Museum der Burgerge- meinde Bern; SVN, Slovenia; CHE, Switzerland; MYS, Malaysia.

0.3 µM of each primer, 0.5 mM dNTPs, and 5 units of Biolase DNA polymerase (Bioline). PCR cycling conditions were as follows: 35 cycles of 30 s at 95 ◦C, 30 s at 48 ◦C, 45 s at 72 ◦C. PCR products were cleaned with ExoSAP-IT (Affymetrix), Sanger sequenced using Big Dyes (Life Technologies) and run on a 3730xl DNA sequencer (Applied Biosystems). Sequences were examined for quality and trimmed to the standard barcode segment (649 bp) using Sequencher 5.01 (Gene Codes). Barcode library While we targeted 649 bp long DNA barcodes we also submitted (Table 1) 18 shorter fragments (>570 bp) that still satisfy the requirements of The Barcode of Life Data System

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 16/25 BOLD systems (Ratnasingham & Hebert, 2007). We combined the 297 species barcodes from this study with publically available Araneae sequences from BOLD retrieved 4 December 2013, for a total of 816 species sequences, which formed the test library for this study. Sequences from BOLD were initially included if the sequence length was at least 600 bases and identification was to species. We further filtered and curated the data to exclude sequences whose identification was anonymous or by non-arachnologists, diverged dramatically from all other spider sequences, or for other reasons the sequences were not deemed to be reliable. After having discarded the above, we did not assess the accuracy of every remaining sequence, as it is well known that both BOLD and GenBank contain errors of various kinds, and we wanted our test library to reflect real world conditions. A single sequence was chosen per species from BOLD using these criteria and added to the original sequences from this project, resulting in 816 species representing 313 genera and 49 families (Table 1 and Table S2). Eighteen sequences were singletons at the family level; the maximum number of species per family was 224. 157 sequences were singletons at the genus level; the maximum number of species per genus was 34. The standalone BLAST+ suite 2.2.28 (Altschul et al., 1990; Zhang et al., 2000) was used to create a custom BLAST database from these sequences. Each sequence was then queried against the full set using blastn (MegaBLAST task, minimum e value of 1e–10, maximum of top ten hits other than the hit of the query to itself). For each hit the percent of identical nucleotides in the aligned region (PIdent) was calculated by BLAST. An advantage of using BLAST is the local nature of the alignment hits returned. This will account for differences in sequence lengths in the dataset, which may otherwise affect pairwise identity calculations of complete alignments. A possible outcome of BLAST results are short aligned regions that have high similarity but omit much of the queried sequence. To investigate this, we compared lengths of aligned regions with query sequence lengths to determine the prevalence of this in this dataset. Custom Python scripts (GitHub https://github.com/mkweskin/spider-blast) were used to parse the results, removing the match of the query to itself and to score whether hits matched the genus and family of the query sequence or not. Obviously, if the generic identification matched, the family identification also matched; families therefore always match more often than genera. On the other hand, singleton generic sequences cannot match correctly at the genus level (for spiders or other poorly known diverse groups), and, likewise, singleton family sequences cannot match correctly at the family level (for spiders or other poorly known diverse groups). We included singletons as targets in order to model more realistically BLAST searches against the BOLD database (many sequences in BOLD are higher level singletons), and also to test more strongly the ability of sequences with two or more species per either genus or family to match correctly. Including 18 singleton family sequences and 157 singleton genus sequences, therefore, increases the probability of misidentification at either ranks and more strongly tests the usefulness of barcodes as supraspecific identification tools. However, because the 18 unique family sequences must fail at both the family and genus levels, and the 157 unique genus level sequences must fail at the genus level, these necessary

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 17/25 Figure 2 Results from the barcode matching test. Frequency distributions of correct and incorrect identifications by percent sequence identity (PIdent) for the top ten and/or best hits at the genus and family level. Shaded areas include hits where no more than 5% of identifications were incorrect.

failures were not included in the overall assessments of the ability of barcode sequences to provide accurate identifications at supraspecific levels.

RESULTS The 816 query sequences returned 8,159 total hits with one query only returning nine hits and all others ten (Table S1). PIdent scores ranged from 75% to 100%. We also examined the length of the sequence matched compared to the entire sequence length. 8,114 hits (>99%) matched to 90% or more of the query sequence length indicating that these results represent matches to large portions of the query validating the use of Percent Sequence Identity in the BLAST hits rather than computing the value for a global alignment between sequences. Figure 2 shows the frequency distributions of PIdent values of correct and incorrect identifications at the genus and family rank. 1. 95% of incorrect genus identifications were below PIdent = 95 when all hits for all queries are included, which suggests the latter value as a heuristic threshold to delimit incorrect from correct identifications (for these data). For only the highest rank hits whose PIdent ≥ 95, 98% of genus identifications were correct. 2. 95% of incorrect family identifications were below PIdent = 91 when all hits for all queries are included, which suggests the latter value as a heuristic threshold to delimit incorrect from correct identifications (for these data). For only the highest rank hits whose PIdent ≥ 91, 97% of family identifications were correct.

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 18/25 3. Library accuracy is crucial, but sequencing, labelling, and identification errors are difficult to detect a priori. The highest ranked incorrect family identification was Meta menardi (Tetragnathidae) to Steatoda grossa (Theridiidae), at PIdent = 96. Further study of the M. menardi sequence shows that the BOLD record is probably a mislabeled Steatoda. The first true incorrect family identification occurs at a PIdent value of 88; the best hit for Octonoba (Uloboridae) is Amaurobius (Amaurobiidae). 4. For the 136 genera with at least two species in the library, 76% (n = 103) best matched congeners. Thirty-three failed, perhaps because sequences were incorrectly identified taxonomically, or the sequence itself may be erroneous, or perhaps due to non- monophyly of genera. 5. The distributions of PIdents for correct family and genus identifications differ significantly from the distributions of incorrect identifications (Fig. 2). 6. Plotted against increasing numbers of species/genus, and genera/family, the proportion of top ten PIdent values that exceed the above suggested threshold values increases. Roughly speaking, 15 species per genus, and 5 genera per family, are sufficient to ensure that best hits represent correct identifications (Fig. 3).

DISCUSSION We show that standard DNA barcodes can accurately assign unknown specimens to genus and family given sufficient sequence identity and sufficient taxonomic representation in the database. Accurate identification (PIdent above which less than 5% of identifications were incorrect) occurred for genera at PIdent values > 95 and families at PIdent values ≥ 91, suggesting these as heuristic thresholds for generic and familial identifications in spiders (shaded in Fig. 2). Accuracy of identification increases with numbers of species/genus and genera/family; above five genera per family and 15 species per genus all identifications were correct (Fig. 3). The accurate identification of specimens remains a critical challenge for megadiverse groups such as arthropods, most other invertebrates, plants, fungi, protists etc. Morphological identification to species, or even more inclusive taxonomic ranks like genera and families, in many cases requires extensive training, and for most groups taxonomic expertise is limited and dwindling—the so called ‘taxonomic impediment’ (Rodman & Cody, 2003; Agnarsson & Kuntner, 2007). DNA barcodes have been proposed as convenient tools to overcome this impediment by making identification a purely technical procedure available to any interested researcher or even ‘citizen scientists.’ However, the accuracy of such a tool strongly depends on the scope and quality of the barcode library (Smit, Reijnen & Stokvis, 2013). Currently available data on databanks like BOLD and GenBank are extensive for some groups, yet the vast majority of species on earth have not yet been barcoded, much less discovered and described taxonomically—each of these tasks is enormous. Even for existing barcoding data, numerous sequences lack accurate taxonomic identification (Collins & Cruickshank, 2013), limiting their utility (e.g., only 58% of Araneae in BOLD are identified to species, and of those many are not correctly identified, as shown in our results; see also Shen, Chen & Murphy, 2013; Blagoev et al., 2016). Therefore, the

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 19/25 Figure 3 Importance of library representation. Relation between proportion of best sequence identity and numbers of species per genus (A), and genera per family (B). Heuristic thresholds to delimit incorrect from correct identifications were 95 and 91 for genus and family, respectively.

identification of unknown specimens through blasting against BOLD or GenBank will be inaccurate if the databases lack close hits or contain errors. While the ideal database would allow species-level identification by containing barcodes from expertly identified and vouchered specimens of all species, we hypothesized that rapid surveys of well-known biotas can help quickly to build valuable tools allowing identification of larger clades such as genera and families. Although we were careful to screen available barcode sequences from BOLD to produce a test library with as few errors as possible, it is certainly possible that errors remained, either due to mistakes in the lab or taxonomic identifications of vouchers. For example, Meta menardi (Tetragnathidae) blasted to Steatoda grossa (Theridiidae) at PIdent = 96, and

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 20/25 BLAST searches on GenBank suggest this Meta sequence is actually a Steatoda. Likewise, the linyphiids Agyneta orites and Incestophantes frigidus sequences were identical; one of these records is probably wrong. These sorts of errors bias identifications and limit utility of barcodes. Other examples of identical barcode sequences were all congeners, and therefore are less likely to involve errors but could indicate faults in taxonomy: Arctosa maculata and A. fulvolineata, Bolyphantes luteolus and B. alticeps, Pardosa alacris and P. trifrons, and Pityohyphantes tacoma and P. cristatus. Likewise, the genus Neriene (Linyphiidae) seems non-monophyletic and identifications were thus not accurate.

CONCLUSIONS These results suggest that accurate assignment of unknown taxa to genus and family is feasible through DNA barcoding. Database quality is crucial. Numbers of potential matches at generic and familial ranks also affect the probability that an unknown sequence will blast best to the correct family or genus. Unlike the inventory of species, biological discovery of family-level clades of life also seems far advanced—few eukaryotic families, apparently, remain to be discovered. Taken together, these results suggest that barcode-targeted sequencing of exemplars from all families of life (and most genera, if possible) should be an important scientific priority. It would enable approximate taxonomic identification of any organism anywhere on Earth by rapid, cheap, purely technical procedures requiring no specialist knowledge—certainly an important milestone in the on-going attempt to discover, classify, and understand the Earth’s biota.

ACKNOWLEDGEMENTS Portions of the laboratory and/or computer work were conducted in and with the support of the L.A.B. facilities of the National Museum of Natural History.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding This work was made possible by a Swiss Contribution to the enlarged EU grant to M Kuntner and C Kropf and the Laboratories of Analytical Biology, National Museum of Natural History, Smithsonian Institution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Grant Disclosures The following grant information was disclosed by the authors: Swiss Contribution to the enlarged EU. Laboratories of Analytical Biology, National Museum of Natural History, Smithsonian Institution. Competing Interests The authors declare there are no competing interests.

Coddington et al. (2016), PeerJ, DOI 10.7717/peerj.2201 21/25 Author Contributions • Jonathan A. Coddington and Ingi Agnarsson conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Ren-Chung Cheng analyzed the data, contributed reagents/materials/analysis tools, reviewed drafts of the paper. • Klemen Čandek and Matjaž Gregorič contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Amy Driskell analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper. • Holger Frick, Rok Kostanjšek, Christian Kropf, Tjaša Lokovšek and Nina Vidergar contributed reagents/materials/analysis tools, reviewed drafts of the paper. • Matthew Kweskin performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Miha Pipan contributed reagents/materials/analysis tools, prepared figures and/or tables, reviewed drafts of the paper. • Matjaž Kuntner conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. DNA Deposition The following information was supplied regarding the deposition of DNA sequences: GenBank accessions are in Table 1 and are also publicly available on BOLD. For all others, see http://ezlab.zrc-sazu.si/dna/. Data Availability The following information was supplied regarding data availability: Github: https://github.com/mkweskin/spider-blast Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.2201#supplemental-information.

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